Analysis of Exosome Uptake May Lead to Simplified Bladder Cancer Diagnosis

A novel cell-cytometry technique has been developed that enables characterization of the uptake and internalization of exosomes by human bladder cancer cells, which may open new routes to the early diagnosis of the disease.

Exosomes, which are 40 to 100 nanometer vesicles secreted by a wide range of mammalian cell types, have been implicated in bladder-cancer cell survival and progression. Exosomes contain various molecular constituents of their cell of origin, including proteins and RNA. Although the exosomal protein composition varies with the cell and tissue of origin, most exosomes contain an evolutionary-conserved common set of protein molecules. The RNA molecules in exosomes include messenger RNA (mRNA) and microRNAs (miRNAs).

Researchers have suggested that shedding of bladder tumor exosomes may promote the progression of bladder lesions. Therefore, a better understanding and characterization of bladder cancer-shed exosome uptake by recipient bladder-cancer cells and their downstream effects are needed.

Several tools are currently used to visualize exosomes and measure their uptake, including Nanosight (Salisbury, United Kingdom) flow cytometers, and confocal microscopes. NanoSight's instruments are based upon a conventional optical microscope fitted with a scientific camera either the LM12 or LM14 viewing unit. Using a laser light source with a wavelength of 405 nm (blue), 532 nm (green), or 638 nm (red), the particles in the sample are illuminated and the scattered light is captured by the camera and displayed on the connected personal computer running proprietary Nanoparticle Tracking Analysis (NTA) software.

There are limitations to analyzing exosomes with any of these methods. Nanoparticle tracking analysis and flow cytometry cannot measure uptake, whereas confocal microscopy is subjective, time consuming, and allows for a limited number of cells to be analyzed.

To correct these shortcomings investigators at Loyola University Medical Center (Maywood, IL, USA) quantitated membrane dye-labeled exosomes isolated from human bladder cancer cells and characterized their uptake by recipient bladder cancer cells using the Amnis Corporation (Seattle, WA, USA) ImageStreamX Imaging Flow Cytometer equipped with INSPIRE software.

Exosomes were isolated by ultracentrifugation from bladder cancer culture conditioned supernatant, labeled with the fluorescent dye PKH-26, and analyzed on the ImageStreamX with an internal standard added to determine concentration.

Results revealed that exosome uptake by recipient bladder-cancer cells was dose and time dependent and that uptake was active and specific and could be partially blocked by heparin treatment.

"Understanding the biology of exosomes could lead to the development of a screening test [for bladder cancer], which would require a simple urine sample," said senior author Dr. Gopal Gupta, assistant professor of urology and surgery at Loyola University Medical Center. "Exosomes derived from a urine sample also might help a physician determine how aggressive the cancer is. This, in turn, could better inform treatment decisions. The test would, in effect, serve as a ‘liquid biopsy,'" said Dr. Gupta.

The paper was published in the January 19, 2014, online edition of the journal BioMed Research International.

Related Links:
Loyola University Medical Center
Amnis Corporation
Nanosight